Phaeobacter inhibens DSM 17395, a member of the Roseobacter clade, was studied for its adaptive strategies to complex and excess nutrient supply, here mimicked by cultivation with Marine Broth (MB). During growth in process-controlled fermenters, P. inhibens DSM 17395 grew faster (3.6-fold higher μmax ) and reached higher optical densities (2.2-fold) with MB medium, as compared to the reference condition of glucose-containing mineral medium. Apparently, in the presence of MB medium, metabolism was tuned to maximize growth rate at the expense of efficiency. Comprehensive proteomic analysis of cells harvested at ½ ODmax identified 1783 (2D DIGE, membrane and extracellular protein-enriched fractions, shotgun) different proteins (50.5% coverage), 315 (based on 2D DIGE) of which displayed differential abundance profiles. Moreover, 145 different metabolites (intra- and extracellular combined) were identified, almost all of which (140) showed abundance changes. During growth with MB medium, P. inhibens DSM 17395 specifically formed the various proteins required for utilization of phospholipids and several amino acids, as well as for gluconeogenesis. Metabolic tuning on amino acid utilization is also reflected by massive discharge of urea to dispose the cell of excess ammonia. Apparently, P. inhibens DSM 17395 modulated its metabolism to simultaneously utilize diverse substrates from the complex nutrient supply.

The denitrifying betaproteobacterium "Aromatoleum aromaticum" strain EbN1 degrades several aromatic compounds, including ethylbenzene, toluene, p-cresol, and phenol, under anoxic conditions. The hydrophobicity of these aromatic solvents determines their toxic properties. Here, we investigated the response of strain EbN1 to aromatic substrates at semi-inhibitory (about 50% growth inhibition) concentrations under two different conditions: first, during anaerobic growth with ethylbenzene (0.32 mM) or toluene (0.74 mM); and second, when anaerobic succinate-utilizing cultures were shocked with ethylbenzene (0.5 mM), toluene (1.2 mM), p-cresol (3.0 mM), and phenol (6.5 mM) as single stressors or as a mixture (total solvent concentration, 2.7 mM). Under all tested conditions impaired growth was paralleled by decelerated nitrate-nitrite consumption. Additionally, alkylbenzene-utilizing cultures accumulated poly(3-hydroxybutyrate) (PHB) up to 10% of the cell dry weight. These physiological responses were also reflected on the proteomic level (as determined by two-dimensional difference gel electrophoresis), e.g., up-regulation of PHB granule-associated phasins, cytochrome cd 1 nitrite reductase of denitrification, and several proteins involved in oxidative (e.g., SodB) and general (e.g., ClpB) stress responses.

Combining omics and enzymatic approaches, catabolic routes of nine selected amino acids (tryptophan, phenylalanine, methionine, leucine, isoleucine, valine, histidine, lysine and threonine) were elucidated in substrate-adapted cells of Phaeobacter inhibens DSM 17395 (displaying conspicuous morphotypes). The catabolic network [excluding tricarboxylic acid (TCA) cycle] was reconstructed from 71 genes (scattered across the chromosome; one-third newly assigned), with 69 encoded proteins and 20 specific metabolites identified, and activities of 10 different enzymes determined. For example, Ph. inhibens DSM 17395 does not degrade lysine via the widespread saccharopine pathway but might rather employ two parallel pathways via 5-aminopentanoate or 2-aminoadipate. Tryptophan degradation proceeds via kynurenine and 2-aminobenzoate; the latter is metabolized as known from Azoarcus evansii. Histidine degradation is analogous to the Pseudomonas-type Hut pathway via N-formyl-l-glutamate. For threonine, only one of the three genome-predicted degradation pathways (employing threonine 3-dehydrogenase) is used. Proteins of the individual peripheral degradation sequences in Ph. inhibens DSM 17395 were apparently substrate-specifically formed contrasting the non-modulated TCA cycle enzymes. Comparison of genes for the reconstructed amino acid degradation network in Ph. inhibens DSM 17395 across 27 other complete genomes of Roseobacter clade members revealed most of them to be widespread among roseobacters.

eThe betaproteobacteria "Aromatoleum aromaticum" pCyN1 and "Thauera" sp. strain pCyN2 anaerobically degrade the plant-derived aromatic hydrocarbon p-cymene (4-isopropyltoluene) under nitrate-reducing conditions. Metabolite analysis of p-cymene-adapted "A. aromaticum" pCyN1 cells demonstrated the specific formation of 4-isopropylbenzyl alcohol and 4-isopropylbenzaldehyde, whereas with "Thauera" sp. pCyN2, exclusively 4-isopropylbenzylsuccinate and tentatively identified (4-isopropylphenyl)itaconate were observed. 4-Isopropylbenzoate in contrast was detected with both strains. Proteogenomic investigation of p-cymene-versus succinate-adapted cells of the two strains revealed distinct protein profiles agreeing with the different metabolites formed from p-cymene. "A. aromaticum" pCyN1 specifically produced (i) a putative p-cymene dehydrogenase (CmdABC) expected to hydroxylate the benzylic methyl group of p-cymene, (ii) two dehydrogenases putatively oxidizing 4-isopropylbenzyl alcohol (Iod) and 4-isopropylbenzaldehyde (Iad), and (iii) the putative 4-isopropylbenzoate-coenzyme A (CoA) ligase (Ibl). The p-cymene-specific protein profile of "Thauera" sp. pCyN2, on the other hand, encompassed proteins homologous to subunits of toluene-activating benzylsuccinate synthase (termed [4-isopropylbenzyl]succinate synthase IbsABCDEF; identified subunits, IbsAE) and protein homologs of the benzylsuccinate ␤-oxidation (Bbs) pathway (termed BisABCDEFGH; all identified except for BisEF). This study reveals that two related denitrifying bacteria employ fundamentally different peripheral degradation routes for one and the same substrate, p-cymene, with the two pathways apparently converging at the level of 4-isopropylbenzoyl-CoA.